The objective of this work is to elucidate the detailed mechanism of control of transcription initiation at the E. coli lac promoter, with particular attention to the cAMP binding gene activating protein CAP. Specific aims include determination of the relationship between DNA sequence and the properties of the CAP- induced DNA bend, along with investigation of the relationship between the magnitude of the induced bend and the intrinsic bending in the naked DNA. We will measure the size of the protein-induced bend, and determine the contribution of writhe to the bend. Experiments that focus on RNA polymerase and transcription will include tracking the CAP-induced bend through the closed, open and initiated complexes. The position, binding stability and reaction kinetics of CAP and polymerase will be examined for promoter mutants with altered CAP sites and bending properties, and with altered phasing between the CAP and polymerase binding sites. Transcription activation in vitro and in vivo will also be studied from these mutants. The interaction of RNA polymerase in the presence of lac repressor will also be examined, and exploratory work on the interaction of a murine homeo domain protein with DNA will be pursued. Methods used are largely a combination of gel electrophoretic separation of protein-DNA complexes, together with chemical and enzymatic characterization of the interactions that stabilize binding.